Compositions and methods for use in a pcr assay for determining the genotype and viral load for respiratory syncytial virus

ABSTRACT

Methods are provided for a sensitive and specific assay for the determination of viral load and genotyping of RSV in a biological sample. Compositions and kits for use in the methods also are provided, including optimized primers for the amplification of and detection of the RSV open reading frames from subtypes A and B, and probes for distinguishing between the subtypes. Also provided are methods for amplifying and sequencing an open reading from of an RSV F protein, and compositions and kits for use in the methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.15/242,856, filed Aug. 22, 2016 (U.S. Pat. No. 10,689,715), which claimspriority to U.S. Provisional Application No. 62/208,556, filed Aug. 21,2015 and U.S. Provisional Application No. 62/244,320, filed Oct. 21,2015. The entire contents of each of these applications are incorporatedherein by reference.

FIELD OF THE INVENTION

Embodiments of the present invention relate to the analysis of arespiratory syncytial virus (“RSV”). In particular, compositions andmethods are provided for determining the genotype and/or viral load forRSV in a biological sample. Compositions and methods for sequencing aRSV fusion protein (F) are also provided.

BACKGROUND

RSV is a negative-sense, single-stranded RNA virus of the paramyxovirusfamily. RSV can cause severe respiratory infections in humans,particularly in infants and children, as well as in the elderly andimmunocompromised. The RSV genome encodes the synthesis of several viralproteins, including three transmembrane glycoproteins (attachmentglycoprotein (G), fusion protein (F), and small hydrophobic protein(SH)); matrix protein M; transcription antitermination protein (M2-1);regulatory protein (M2-2); three proteins associated with thenucleocapsid (N, P, and L); and two nonstructural proteins (NS1 andNS2). RSV strains may be separated into two main groups, genotypes A andB (RSV A and RSV B).

Human RSV (hRSV) is the major cause of severe respiratory infectionssuch as bronchiolitis and lower tract illness affecting mostly newbornsand young children. Approximately 30 million children younger than fiveyears old suffer from acute lower respiratory infection due to hRSV, andhRSV causes numerous complications in premature born patients as well asinfants suffering from congenital heart disease and immune deficiency.Long-term effects of RSV infection include central nervous systemalterations, seizures, central apnea, and encephalopathy, to name a few.Over 200,000 deaths per year can be attributed to hRSV and there are noefficient therapies to counteract the disease. Therefore, efforts arefocused on generating a vaccine to prevent hRSV infection as well asdeveloping new therapeutic drugs to treat RSV infection and reduce thepotential long-term effects caused by RSV infection.

In order for health care providers to determine the effectiveness oftreatment for a particular individual or to determine the best course oftreatment for a particular individual, it is important to determine theamount of RSV present in the individual and/or the genotype of RSV thathas infected the individual. What is needed, therefore, are compositionsand methods for the efficient genotyping of an RSV and for the efficientdetermination of viral load.

SUMMARY

Provided herein are methods for simultaneously determining the viralload and genotype of a respiratory syncytial virus (RSV) in a biologicalsample. These methods may include the steps of a) amplifying a nucleicacid sequence encoding an RSV open reading frame (ORF) in a biologicalsample using a set of primers that specifically amplifies an RSV subtypeA ORF and/or an RSV subtype B ORF and at least two nucleic acid probesin a quantitative polymerase chain reaction assay, wherein at least oneprobe specifically binds to an RSV subtype A nucleic acid sequence andat least one probe specifically binds to an RSV subtype B nucleic acidsequence, and wherein the at least two nucleic acid probes aredifferentially labeled; and b) determining the viral load and the RSVsubtype(s) present in the biological sample.

Methods are provided for determining the genotype or viral load of arespiratory syncytial virus (RSV) in a biological sample. The methodsmay include the steps of a) amplifying a nucleic acid sequence encodingan RSV open reading frame (ORF) in a biological sample using a primerthat comprises SEQ ID NO:1, a primer that comprises SEQ ID NO:2, a probethat comprises SEQ ID NO:3, and a probe that comprises SEQ ID NO:4 in aquantitative polymerase chain reaction assay, wherein the probes aredifferentially labeled; and b) determining the presence or absence of anamplification product that binds one of the probes, or determining theviral load, thereby determining the RSV genotype(s) present and/or viralload of the RSV in the biological sample. In addition, methods areprovided for determining the genotype or viral load of a respiratorysyncytial virus (RSV) in a biological sample, including the steps of a)amplifying a nucleic acid sequence encoding an RSV open reading frame(ORF) in a biological sample using a primer that comprises SEQ ID NO:1,a primer that comprises SEQ ID NO:2, and either a probe that comprisesSEQ ID NO:3 or a probe that comprises SEQ ID NO:4 in a quantitativepolymerase chain reaction assay, wherein the probe is labeled; and b)determining the presence or absence of an amplification product thatbinds the probe, or determining the viral load, thereby determining theRSV genotype(s) present or viral load of the RSV in the biologicalsample.

Also provided are methods for diagnosing an RSV infection in a subject.These methods may include the steps of a) amplifying a nucleic acidsequence encoding an RSV ORF in a biological sample from the subjectusing a set of primers that specifically amplifies an RSV subtype A ORFand/or an RSV subtype B ORF and at least two nucleic acid probes in aquantitative polymerase chain reaction assay, wherein at least one probespecifically binds to an RSV subtype A nucleic acid sequence and atleast one probe specifically binds to an RSV subtype B nucleic acidsequence, and wherein the at least two nucleic acid probes aredifferentially labeled; and b) determining the viral load and the RSVsubtype(s) present in the biological sample, thereby diagnosing asubject with an RSV infection.

Methods for determining the efficacy of a therapy for RSV infection in asubject also are provided. These methods may include the steps ofamplifying a nucleic acid sequence encoding an RSV ORF in a firstbiological sample from the subject using a set of primers thatspecifically amplifies an RSV subtype A ORF and/or an RSV B ORF and atleast two nucleic acid probes in a quantitative polymerase chainreaction assay, wherein at least one probe specifically binds to an RSVsubtype A nucleic acid sequence and at least one probe specificallybinds to an RSV subtype B nucleic acid sequence, and wherein the atleast two nucleic acid probes are differentially labeled; b) determiningthe viral load and the RSV subtype(s) present in the first biologicalsample; c) amplifying a nucleic acid sequence encoding an RSV ORF in asecond biological sample from the subject using a set of primers thatspecifically amplifies an RSV subtype A ORF and/or an RSV subtype B ORFand at least two nucleic acid probes in a quantitative polymerase chainreaction assay, wherein the subject has undergone at least one treatmentwith a first therapy for RSV infection, wherein at least one probespecifically binds to an RSV subtype A nucleic acid sequence and atleast one probe specifically binds to an RSV subtype B nucleic acidsequence, and wherein the at least two nucleic acid probes aredifferentially labeled; d) determining the viral load and the RSVsubtype(s) present in the second biological sample; comparing the viralload and/or RSV subtypes of the first biological sample with the viralload and/or RSV subtypes of the second biological sample, wherein adecrease in viral load in the second biological sample or a change inRSV subtype(s) as compared to the viral load and/or subtypes in thefirst biological sample indicates that the treatment is effective andwherein an increase or no change in viral load and/or RSV subtype(s) orno change in the subtype(s) as compared to the viral load and/or RSVsubtype (s) in the first biological sample indicates that the treatmentis ineffective.

Nucleic acid probes for the detection of the genotype of an RSV areprovided. In some embodiments, the probes comprise SEQ ID NO:3 or SEQ IDNO:4. In other embodiments, the probes comprise SEQ ID NO:36(GCAAGCTTAACAACTGAAATTCAAATCAAC) or SEQ ID NO:37(TCAAGCTTGACATCAGAAATACAAGTCAAT). Methods are provided for using one ormore of these probes for the detection of the genotype of an RSV in abiological sample. In some embodiments, a control probe is used. Thecontrol probe may comprise, for example, SEQ ID NO:5.

Kits are provided for the detection of viral load and the genotype of anRSV in a biological sample. The kits may include a nucleic acid primercomprising SEQ ID NO:1, a nucleic acid primer comprising SEQ ID NO:2, aprobe comprising SEQ ID NO:3, and a probe comprising SEQ ID NO:4. Incertain embodiments, the kits further comprise a nucleic acid primercomprising SEQ ID NO:34, a nucleic acid primer comprising SEQ ID NO:35,a nucleic acid probe comprising SEQ ID NO:36 and a nucleic acid probecomprising SEQ ID NO:37.

Also provided are methods for amplifying and sequencing the open readingframe (ORF) of an RSV F protein and methods for amplifying andsequencing the ORF of an RSV F protein. Certain of these methods mayinclude the step of amplifying a nucleic acid sequence encoding an RSV Fopen reading frame in a biological sample using a set of primerscomprising SEQ ID NO:6 and SEQ ID NO:7 in a polymerase chain reactionassay. In some embodiments, the methods further may include sequencingthe amplification product using one or more primers selected from thegroup consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, and SEQ ID NO:19. Certain other methods may include the step ofamplifying a nucleic acid sequence encoding an RSV F open reading framein a biological sample using a set of primers comprising SEQ ID NO:8 andSEQ ID NO:9. In some embodiments, the methods may further includesequencing the amplification product in a polymerase chain reactionassay using one or more sequencing primers selected from the groupconsisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 andSEQ ID NO:29.

Further provided are kits for the amplification and sequencing the ORFof an RSV F protein and kits for the amplification and sequencing theORF of an RSV F protein. The kits include a set of primers comprisingSEQ ID NO:6 and SEQ ID NO:7 and/or a set of primers comprising SEQ IDNO:8 and SEQ ID NO:9. In some embodiments, the kits may include one ormore primers selected from the group consisting of SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29

DESCRIPTION OF THE DRAWINGS

The figures provided herein are intended to illustrate certainembodiments and/or features of the compositions and methods, and tosupplement any description(s) of the compositions and methods. Thefigures do not limit the scope of the compositions and methods, unlessthe written description expressly indicates that such is the case.

FIGS. 1A and 1B show the results of multiplex qRT-PCR assays tosimultaneously determine viral load and subtype specificity of RSV. FIG.1A is a graphical representation of the data shown in the table in FIG.1B, with RSV subtype A data shown in the top panels and RSV subtype Bdata shown in the bottom panels.

FIG. 2 is a table showing the results of a comparative study of theqRT-PCR assay with GenMark Diagnostics RVP qualitative assay. The datashown represent the results of qRT-PCR reactions being performed intriplex.

FIG. 3 presents tables showing a proficiency panel for the multiplexqRT-PCR assay. The data are the result of 10-fold serial dilutions,triplicate samples of known viral load, reported in copies/mL. Known VLrefers to the viral load as detected using a singleplex assay, and MGB3p1ex refers to the multiplex qRT-PCR assay in which RSV subtype A, RSVsubtype B, and control may be detected in the same assay. The datademonstrates that there is no loss in sensitivity in the multiplexedreaction.

FIG. 4 is a schematic diagram of the cloning vector pCR®4-TOPO and thecloning site for the amplification product produced using universalprimers that amplify the N region of RSV A2 and RSV B1.

FIG. 5 shows the input and output screens for the online calculator usedto determine copy number for the RSV virus.

FIG. 6A is a quality control plot for Illumina MiSeq® sequencing of anhRSV isolate, showing that the Q-score is greater than 30 across reads.FIG. 6B is a quality control plot showing that greater than 99% of readsmapped to the reference genome with evenly distributed read and insertlengths. Minor variants were detected using 3-10% thresholds, withaccompanying data on each variant codon, amino acid, and frequency.

FIG. 7A is a schematic diagram showing the relative locations of thesequencing primers used to sequence the RSV F ORF using Sangersequencing. FIG. 7B is a neighbor joining tree of the RSV A and RSV Bsamples derived from Illumina MiSeq® analysis.

DESCRIPTION

Quantitative multiplex reverse transcriptase polymerase chain reaction(qRT-PCR) assay methods and compositions for use in these methods areprovided. The present methods provide a sensitive and specific assay forthe determination of viral load, genotyping of RSV, or both in abiological sample.

In certain embodiments, the methods use universal RSV amplificationprimers to amplify the desired sequence from both RSV subtypes A and Bin the presence of differentially labeled RSV subtype A specific probesand RSV subtype B specific probes that are used to distinguish betweeneach RSV strain. These qRT-PCR methods may be multiplex assays employinguniversal RSV amplification primers and differentially labeled RSVsubtype A and RSV subtype B specific probes. These methods allow for thequantification of RSV viral load and the determination of the RSVsubtype (A or B).

For example, provided herein are methods for simultaneously determiningthe viral load and genotype of a respiratory syncytial virus (RSV) in abiological sample. The methods include the steps of a) amplifying anucleic acid sequence encoding an RSV open reading frame (ORF) in abiological sample using a set of primers that specifically amplifies anRSV subtype A ORF and/or an RSV subtype B ORF and at least two nucleicacid probes in a quantitative polymerase chain reaction assay, whereinat least one probe specifically binds to an RSV subtype A nucleic acidsequence and at least one probe specifically binds to an RSV subtype Bnucleic acid sequence, and wherein the at least two nucleic acid probesare differentially labeled; and b) determining the viral load and theRSV subtype(s) present in the biological sample. Alternatively, methodsare provided for determining the genotype or viral load of a respiratorysyncytial virus (RSV) in a biological sample, including the steps of a)amplifying a nucleic acid sequence encoding an RSV open reading frame(ORF) in a biological sample using a primer that comprises SEQ ID NO:1,a primer that comprises SEQ ID NO:2, a probe that comprises SEQ ID NO:3,and a probe that comprises SEQ ID NO:4 in a quantitative polymerasechain reaction assay, wherein the probes are differentially labeled; andb) determining the presence or absence of an amplification product thatbinds one of the probes, or determining the viral load, therebydetermining the RSV genotype(s) present or viral load of the RSV in thebiological sample. These methods also may be used for diagnosing asubject with RSV infection or for determining the efficacy of atreatment or vaccine for RSV.

As used herein, “a set of universal primers” is a set of nucleic acidprimers that can specifically amplify an RSV subtype A (RSV A) nucleicacid and/or RSV subtype B (RSV B) nucleic acid, if an RSV A and/or anRSV B nucleic acid molecule is present in the biological sample. In themethods provided herein, the set of universal primers can include a pairof nucleic acid primers that specifically amplify a region of the RSVnucleocapsid (N) protein in RSV A and/or RSV B. Optionally, the set ofuniversal primers that specifically amplify a region of the RSVnucleocapsid (N) protein in RSV A and/or RSV B includes a nucleic acidsequence comprising SEQ ID NO:1 and a nucleic acid sequence comprisingSEQ ID NO:2. Other primers that can be used include, but are not limitedto a nucleic acid comprising SEQ ID NO:34 (RGAAATGAAATTYGAAGTRTTAAC) anda nucleic acid comprising SEQ ID NO:35

(GARTCATGCCTRTATTCTGGAGC).

As used herein, the term “primer” refers to an oligonucleotide that caninitiate 5′ to 3′ synthesis of a primer extension product that iscomplementary to a nucleic acid strand. In the methods provided herein,the set of primers contains a forward primer and a reverse primer.

As used herein, the term “probe” refers to an oligonucleotide that formsa hybrid structure with a target sequence, for example, an RSV A or anRSV B sequence, contained in a biological sample, due to complementarityof at least one sequence in the probe with the target sequence. Thenucleotides of any particular probe can be ribonucleotides,deoxyribonucleotides, and/or synthetic nucleotide analogs. Examples ofnucleic acid probes that can be used in the methods provided hereininclude, but are not limited to, a nucleic acid probe comprising SEQ IDNO:3 or SEQ ID NO:36 (GCAAGCTTAACAACTGAAATTCAAATCA) that specificallybinds to an RSV subtype A nucleic acid sequence and a nucleic acid probecomprising SEQ ID NO:4 or SEQ ID NO:37 (TCAAGCTTGACATCAGAAATACAAGTCAAT)that specifically binds to an RSV subtype B nucleic acid sequence.

As used herein, the term “complementary” or “complementarity” refers tobase pairing between nucleotides or nucleic acids. Examples include basepairing between an oligonucleotide primer and a primer binding site on asingle-stranded nucleic acid to be sequenced or amplified and basepairing between a probe and a target nucleic acid sequence.Complementary nucleotides are, generally, adenine (A) and thymine (T)(or A and uracil (U)), and guanine (G) and cytosine (C). It isunderstood that the specific nucleotide sequence lengths provided hereinare exemplary and not limiting. Further, sequences covering the samepositions or locations within a RSV sequence, for example, positions ofthe nucleotide sequence encoding the RSV N protein or the RSV F protein,but having a fewer or greater number of bases as compared to the primeror probe sequences provided herein, are also contemplated hereinprovided that the sequences hybridize to the same locations on thetarget RSV A and/or RSV B sequence as the disclosed sequences. Those ofskill in the art will appreciate that nucleic acids do not require 100%complementarity in order to hybridize. Therefore, probe and primersequences that have about 80%, about 85%, about 90%, or about 95%identity with the probe and primer sequences provided herein can also beused in the methods described herein.

In the methods set forth herein, numerous methods are available in theart for determining viral load. For example, and not to be limiting,qRT-PCR can be used to quantify the amount of RSV virus, i.e., viralload, viral burden, or viral titer, in a biological sample. qRT-PCRmethods for determining viral load are known in the art. (See forexample, Payungporn et al. “Single step multiplex real-time RT-PCR forH5N 1 influenza A virus detection.” J Virol Methods. Sep. 22, 2005;Landolt et la. “Use of real-time reverse transcriptase polymerase chainreaction assay and cell culture methods for detection of swine influenzaA viruses” Am J Vet Res. 2005 January; 66(1):119-24). Those of skill inthe art understand that RT-PCR assesses the kinetics of amplification,which is based on the starting template copy number. The metric is Ct,which stands for cycle threshold. Ct is a relative measure of theconcentration of target in the PCR reaction that represents the computedamplification cycle at which point the detection of released fluorophorestatistically exceeds the background fluorophore signal (the inflectionpoint of fluorophore signal generation). In other words, Ct is thenumber of cycles required for the fluorescent signal to cross thethreshold, i.e., exceed background level. One of skill in the art wouldknow how to establish Ct cut-off values for positivity as well asgenerate standard curves for viral load quantitation. Other methodsinclude, but are not limited to digital PCR and transcriptionloop-mediated isothermal amplification (LAMP). These and other methodsdeveloped in the future for amplification and detection of nucleic acidscan be used in the methods provided herein.

In the methods set forth herein, the RSV sequences in the biologicalsample can be reverse transcribed prior to amplification. Alternatively,the RSV sequences in the biological sample can be reverse transcribedand amplified in a single step.

By using differentially labeled probes for RSV A and RSV B, theproduction of amplification products during each cycle of the PCRreaction can be monitored, thus allowing discrimination between multipleviral genotypes and quantification of each genotype. In the methodsdescribed herein, the probes and/or primers can be labeled. Labelsinclude atoms and molecules that are attached to a nucleic acid in orderto produce a signal that is detectable and quantifiable by fluorescence,chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-raydiffraction, absorption, magnetism, enzymatic activity, and the like.Examples of labels include fluorophores, chromophores, radioactiveatoms, enzymes, and ligands having specific binding partners.

For example, and not to be limiting, probes can be labeled with donorand corresponding acceptor fluorescent moieties. Examples of fluorescentdonor moieties include, but are not limited to FAM™, VIC® and NED™fluorescent dyes. Examples of fluorescent acceptor moieties include, butare not limited to, BHQ® and ZEN™. These and other fluorescent moietiesfor use in RT-PCR are known to those of skill in the art and areavailable from numerous commercial sources.

As set forth above, the methods provided herein can optionally includeamplification and detection of a recovery control. For example, themethods provided herein can include a set of primers that specificallyamplifies a bacteriophage MS2 region and a nucleic acid probe thatspecifically binds to a bacteriophage M2 nucleic acid. Examples ofprimers that can be used to amplify an MS2 region include a nucleic acidsequence comprising SEQ ID NO:30 and a nucleic acid sequence comprisingSEQ ID NO:31. An example of a probe that can be used to detect an MS2nucleic acid is set forth herein as SEQ ID NO:5.

A recovery control can include a nucleic acid molecule from a differentsource other than an RSV molecule. For example, a recovery control caninclude a nucleic acid from a different virus or bacteriophage, such asMS2. One of skill in the art would know how to design primers thatspecifically amplify a control amplification product and probes thatspecifically detect the recovery control amplification product. Themethods provided herein also may include separate samples that includeone or more positive control samples that include RSV A and/or RSV Bsequences.

Any of the methods of determining viral load and/or RSV subtype(s)described herein can further comprise amplifying the RSV F open readingframe in a biological sample and b) sequencing the amplificationproduct. Methods, primers for amplification, and primers for sequencingof RSV A and RSV B F open reading frames are set forth below. Thus,sequencing of the RSV F protein can be used to confirm the subtypesidentified in the methods provided herein.

As used herein, a biological sample is a sample derived from a subjectsuch as a mammal or human and includes, but is not limited to, anybiological fluid, including a bodily fluid. Examples of bodily fluidsinclude, but are not limited to, whole blood, plasma, serum, urine,saliva, ocular fluid, ascites, sputum, throat swabs, throat washings,nasal swabs, lower respiratory tract specimens, a stool sample, spinalfluid, tissue infiltrate, pleural effusions, lung lavage fluid, and thelike. The biological fluid includes a cell culture medium or supernatantof cultured cells from the subject.

As used throughout, the term “subject” refers to an individual.Preferably, the subject is a mammal such as a primate, and, morepreferably, a human of any age, including a newborn or a child.Non-human primates are subjects as well. The term subject includesdomesticated animals, such as cats, dogs, etc., livestock (for example,cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (forexample, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig,etc.). Thus, veterinary uses are contemplated herein.

Also provided are methods for diagnosing an RSV infection in a subject.The methods include the steps of a) amplifying a nucleic acid sequenceencoding an RSV ORF in a biological sample using a set of primers thatspecifically amplifies an RSV subtype A ORF and/or an RSV subtype B ORFand at least two nucleic acid probes in a quantitative polymerase chainreaction assay, wherein at least one probe specifically binds to an RSVsubtype A nucleic acid sequence and at least one probe specificallybinds to an RSV subtype B nucleic acid sequence, and wherein the atleast two nucleic acid probes are differentially labeled; and b)determining the viral load and the RSV subtype(s) present in thebiological sample, thereby diagnosing a subject with an RSV infection.

The methods of diagnosing a subject with an RSV infection can furthercomprise treating the subject for an RSV infection. Antiviral drugs suchas ribavirin, motavizumab, palivizumab (Synagis®), GS1, GS-5806 (SeeJordan et al. “Antiviral Efficacy of Respiratory Syncytial Virus (RSV)Fusion Inhibitor in a Bovine Model of RSV Infection,” 59 (8): 4889-4900(2015) which is hereby incorporated in its entirety by this reference,particularly as it relates to the structures and activities of GS1 andGS-5806.), VP14637 and JNJ-2408068 (See Douglas et al. “Small MoleculesVP-1637 and JNJ-2408068 Inhibit Respiratory Syncytial Virus Fusion bySimilar Mechanisms,” 49(6):2460-2466 (2005), which is herebyincorporated in its entirety by this reference, particularly as itrelates to for the structures and activities of VP14637 and JNJ-2408068)can be used to treat RSV infections. As used herein, the termstreatment, treat, or treating refers to a method of reducing the effectsof a disease or condition or symptom of the disease or condition. Thus,in the disclosed methods, treatment can refer to an about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease or condition or symptom of the disease or condition.For example, a method for treating a disease is considered to be atreatment if there is an about 10% reduction in one or more symptoms ofthe disease in a subject as compared to a control. A control subject canbe a subject that has not been treated for an RSV infection. Thus, thereduction can be an about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or any percent reduction in between about 10% and 100% as comparedto native or control levels. It is understood that treatment does notnecessarily refer to a cure or complete ablation of the disease,condition, or symptoms of the disease or condition.

Further provided are methods for determining the efficacy of a therapyfor RSV infection in a subject. The methods may include a) amplifying anucleic acid sequence encoding an RSV ORF in a first biological samplefrom the subject using a set of primers that specifically amplifies anRSV subtype A ORF and/or an RSV subtype B ORF and at least two nucleicacid probes in a quantitative polymerase chain reaction assay, whereinat least one probe specifically binds to an RSV subtype A nucleic acidsequence and at least one probe specifically binds to an RSV subtype Bnucleic acid sequence, and wherein the at least two nucleic acid probesare differentially labeled; b) determining the viral load and the RSVsubtype(s) present in the first biological sample; c) amplifying anucleic acid sequence encoding an RSV ORF in a second biological samplefrom the subject using a set of primers that specifically amplifies anRSV subtype A ORF and/or an RSV subtype B ORF and at least two nucleicacid probes in a quantitative polymerase chain reaction assay, whereinthe subject has undergone at least one treatment with a first therapyfor RSV infection, wherein at least one probe specifically binds to anRSV subtype A nucleic acid sequence and at least one probe specificallybinds to an RSV subtype B nucleic acid sequence, and wherein the atleast two nucleic acid probes are differentially labeled; d) determiningthe viral load and the RSV subtype(s) present in the second biologicalsample; e) comparing the viral load and/or RSV subtypes of the firstbiological sample with the viral load and/or RSV subtypes of the secondbiological sample, wherein a decrease in viral load and/or a change inthe subtype(s) in the second biological sample as compared to the viralload and/or the subtype(s) in the first biological sample indicates thetreatment is effective and wherein an increase or no change in viralload and/or no change in the subtype(s) as compared to the viral loadand/or subtype (s) in the first biological sample indicates thetreatment is ineffective.

The methods of determining the efficacy of a therapy can also be usedduring treatment. For example, a first biological sample can be obtainedfrom the subject after the first treatment for an RSV infection and asecond biological sample can be obtained from the subject after thesecond treatment for an RSV infection in order to determine thedifference in viral load and/or the amount of an RSV subtype(s), if any,between the first and second biological sample. Similarly, a firstbiological sample can be obtained from the subject after the secondtreatment for an RSV infection and a second biological sample can beobtained from the subject after the third treatment for an RSV infectionin order to determine the difference in viral load and/or the amount ofan RSV subtype(s), if any, between the second and third biologicalsample.

The decrease in viral load does not have to be complete as this decreasecan be about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in viral load. The decrease in viral load can also be a logiodecrease, for example, a 3-log decrease, a 4-log decrease, a 5-logdecrease, or greater. If there is an increase or no change in viral loadthe type, dosage and/or frequency of the therapy can be modified for thesubject.

Further provided are methods for determining the efficacy of an RSVvaccine in a subject. The methods include a) administering an RSVvaccine to a subject; b) contacting the subject with an RSV virus; b)amplifying a nucleic acid sequence encoding an RSV ORF in a biologicalsample from the vaccinated subject using a set of primers thatspecifically amplifies an RSV subtype A ORF and/or an RSV subtype B ORFand at least two nucleic acid probes in a quantitative polymerase chainreaction assay, wherein at least one probe specifically binds to an RSVsubtype A nucleic acid sequence and at least one probe specificallybinds to an RSV subtype B nucleic acid sequence, and wherein the atleast two nucleic acid probes are differentially labeled; c) determiningthe viral load and the RSV subtype(s) present in the biological sample;and d) comparing the viral load and/or RSV subtypes of the biologicalsample with the viral load and/or RSV subtypes of a control sample,wherein the control sample is from a subject that was contacted with theRSV virus and was not vaccinated with the RSV vaccine, wherein adecrease in viral load and/or a change in the subtype(s) in thebiological sample as compared to the viral load and/or the subtype(s) inthe control sample indicates the vaccine is protective and wherein anincrease or no change in viral load and/or no change in the subtype(s)as compared to the viral load and/or subtype (s) in the first biologicalsample indicates the vaccine is not protective.

Protection does not have to be complete as the decrease in viral loadcan be about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in viral load. The decrease in viral load can also be a logiodecrease, for example, a 3-log decrease, a 4-log decrease, a 5-logdecrease, or greater.

The methods of determining the efficacy of a therapy for RSV infectionor the efficacy of an RSV vaccine can further comprise the steps ofdetermining the sequence of the RSV F open reading frame from thebiological sample before treatment, determining the sequence of the RSVF open reading frame after treatment and comparing the sequences. Ifchanges in the sequence occur after treatment, for example, mutationsassociated with resistance to a particular therapy, this would indicatethat the subject could develop resistance to the therapy. These changesin the F protein sequence can be monitored in the subject as treatmentprogresses. The type, dosage, and/or frequency of the therapy can bemodified for the subject based on changes in the F protein nucleic acidsequence.

Sequencing of RSV Isolates

Methods for sequencing RSV isolates are provided. More specifically,provided herein are methods for amplifying and sequencing the openreading frame (ORF) of the RSV F protein from RSV subtype A and/or RSVsubtype B. Certain methods include the step of a) amplifying a nucleicacid sequence encoding an RSV F open reading frame in a biologicalsample using a set of primers comprising SEQ ID NO:6 and SEQ ID NO:7. Insome embodiments of these methods, the methods further include the stepof b) sequencing the amplification product using one or more primersselected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19. Cycle sequencing is method in whichsuccessive rounds of PCR (denaturation, annealing and extension) in athermal cycler result in linear amplification of extension products thatare then loaded onto a gel or injected into a capillary for sequenceanalysis. As set forth in the Examples, the RSV F open reading frame isreverse transcribed prior to amplification. After amplification of theRSV F open reading frame, full-length sequencing may be performed withten overlapping primers per subtype.

Also provided are methods for amplifying and sequencing the open readingframe (ORF) of the RSV F protein including the step of a) amplifying anucleic acid sequence encoding an RSV F open reading frame in abiological sample using a set of primers comprising SEQ ID NO:8 and SEQID NO:9. In some embodiments of these methods, the methods furtherinclude the step of b) sequencing the amplification product using one ormore primers selected from the group consisting of SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. As set forth in theExamples, the RSV F open reading frame is reverse transcribed prior toamplification. After amplification of the RSV F open reading frame,full-length sequencing may be performed with ten overlapping primers persubtype.

In the sequencing methods provided herein, the amplification product canbe sequenced using any method, including but not limited to, cyclesequencing, Maxam-Gilbert sequencing, Sanger sequencing, next generationsequencing, and the like. Therefore, in some embodiments, for example, anucleic acid sequence encoding an RSV F open reading frame in abiological sample is amplified using a set of primers comprising SEQ IDNO:6 and SEQ ID NO:7 or a set of primers comprising SEQ ID NO:8 and SEQID NO:9, and the amplification product is sequenced by next generationsequencing analysis.

Probes and Kits

Nucleic acid probes for the detection of the genotype of an RSV areprovided. In some embodiments, the probes comprise SEQ ID NO:3 or SEQ IDNO:4. In other embodiments, the probes comprise SEQ ID NO:36 or SEQ IDNO:37. Methods are provided for using one or more of these probes forthe detection of the genotype of an RSV in a biological sample.

Also provided is a kit for the detection of viral load and the genotypeof an RSV in a biological sample comprising a set of nucleic acidprimers that specifically amplifies the N region of the RSV A subtype(RSV A) and the RSV B subtype (RSV B), a nucleic acid probe thatspecifically binds to an RSV A nucleic acid and a nucleic acid probethat specifically binds to an RSV B nucleic acid. For example, the kitcan include a nucleic acid primer comprising SEQ ID NO:1 and a nucleicacid primer comprising SEQ ID. NO: 2 for amplification of the N regionof RSV A and the RSV B subtypes as well as a probe comprising SEQ IDNO:3 that specifically binds to RSV A nucleic acid sequence and a probecomprising SEQ ID NO:4 that specifically binds to a RSV B nucleic acidsequence. In another embodiment, the kit can include a nucleic acidprimer comprising SEQ ID NO:34 and a nucleic acid primer comprising SEQID NO:35 for amplification of the N region of RSV A and the RSV Bsubtypes as well as a probe comprising SEQ ID NO:36 that specificallybinds to RSV A nucleic acid sequence and a probe comprising SEQ ID NO:37that specifically binds to a RSV B nucleic acid sequence. In someembodiments, the kit further includes a primer comprising SEQ ID NO:30,a nucleic acid primer comprising SEQ ID NO:31, and a nucleic acid probecomprising SEQ ID NO:5. Optionally, one or more of the primers and/orprobes can be labeled with different, detectable labels.

Also provided is a kit for the amplification and sequencing of an RSV FORF. The kit includes an amplification primer comprising SEQ ID NO:6 andan amplification primer comprising SEQ ID NO:7. The kit may furtherinclude one or more primers selected from the group consisting of SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19.Optionally, one or more of the primers can be labeled with the same ordifferent detectable labels. Optionally, the kit further comprisesreagents for isolating and/or sequencing the DNA in the sample.

Further provided is a kit for the amplification and sequencing of an RSVF ORF. The kit includes an amplification primer comprising SEQ ID NO:8and an amplification primer comprising SEQ ID NO:9. The kit may furtherinclude one or more primers selected from the group consisting of SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.Optionally, one or more of the primers can be labeled with the same ordifferent detectable labels. Optionally, the kit further comprisesreagents for isolating and/or sequencing the DNA in the sample.

Disclosed are materials and compositions that can be used for, can beused in conjunction with, can be used in preparation for, or areproducts of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

EXAMPLES

The following non-limiting examples are intended to illustrate certainembodiments and/or features of the compositions and methods and tosupplement any description(s) of the compositions and methods.

Example 1 Total Nucleic Acid Purification

The nucleic acids were purified from RSV from biological samples usingthe Biomerieux NucliSENS easyMAG® system. The materials for nucleic acidpurification included the following:

NucliSENS easyMag instrument

easyMag Wash Buffers 1, 2, 3

easyMag Lysis Buffer

easyMag Magnetic Silica

Respiratory samples (˜200 μl)

The nucleic acid purification was performed as follows. Twenty-twosamples were equilibrated to room temperature under a laminar flow hood.The NucliSENS easyMAG unit was turned on before logging into thesoftware. A series was prepared as follows: the sample type was selected(other), the extraction protocol was selected (Generic 2.0.1), thesample volume was selected (200 μl), the elution volume of each samplewas selected (50 μl), and the type of lysis was selected (on-board).

Samples to be processed were identified. The first icon to the right wasclicked in order to choose whether incubation lysis must be performed bythe instrument NucliSens easyMAG or outside (‘on board’ or ‘off board’).For the incubation with silica, ‘on board’ was selected. All sampleswere selected and ‘Add selected samples to run’ was selected. Thebarcode of the reagents was entered with the reader by first clicking onthe barcode of the machine and then on the bottle. Then 200 μl of samplewere placed in each well of a disposable (8-well plastic cartridge) inthe laminar flow hood. Three disposables and their suction combs wereinstalled on the instrument by first scanning the bar code of theposition and then the bar code of the disposable. Ten μl of internalcontrol MS2 was added per well of disposable. Delivery of lysis bufferwas initiated.

The extraction protocol for off-board lysis was performed using theNucliSens easyMAG Total Nucleic Acid Extraction standard protocol. Dryswabs were re-suspended by adding 1,500 μl of the suspension buffer(easyMAG Lysis Buffer, UTM, saline solution, AVE buffer, or RNAlater).All samples were vortexed vigorously for 15 seconds at least threetimes. Some of the supernatant was used for nucleic acid extraction. Theremaining supernatant was transferred to a 2 mL microcentrifuge tube forlong term storage. The sample input volume for nucleic acid extractionwas 500 μl with an elution output of 100 μl. During the lysis stage, 550μl of RNAse-free ddH2O was combined with 550 μl Magnetic Silica andmixed thoroughly. Then 125 μl of silica was added per well and mixedwith a multichannel pipette. The extraction procedure was launched, andthe extracted nucleic acids were collected in the half-hour after theend of the run. The extracted nucleic acids were transferred to sterile1.5 ml tubes used directly or stored at −80° C.

Example 2 Determination of Consensus Sequence for the RSV N Open ReadingFrame

Consensus sequences based on the top 100 hits for an RSV A2 and B1 N(open reading frame) ORF reference sequences were obtained. Bothconsensus sequences are set forth below.

RSV A2 N ORF consensus (SEQ ID NO: 32)ATGGCTCTTAGCAAAGTCAAGTTGAAYGATACACTCAACAAAGATCAACTTCTGTCATCCAGCAAATACACCATCCAACGGAGCACAGGAGAYAGYATTGAYACTCCTAATTATGATGTGCAGAAACACATCAAYAAGYTATGTGGCATGTTATTAATCACAGAAGATGCTAATCATAAATTCACTGGGKTAATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCARAGATGCDGGATATCATGTAAAAGCWAATGGAGTGGATGTAACAACACATCGTCAAGAYATTAATGGRAAAGAAATGAAATTTGAAGTGTTAACATTRGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGRAAATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTRGCTCCAGAATACAGGCATGACTCWCCTGATTGTGGRATGATAATATTATGTATAGCRGCATTAGTAATAACCAAATTAGCAGCAGGGGATAGATCTGGTCTTACAGCYGTRATTAGGAGAGCTAATAATGTYCTAAAAAATGAAATGAAACGTTATAAAGGCTTACTACCHAAGGATATAGCHAACAGYTTCTATGAAGTGTTTGAAAAATATCCTCACTTTATAGATGTTTTTGTTCATTTTGGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGAATGCCTATGGTGCAGGGCAAGTGATGTTACGGTGGGRGTCTTAGCAAAATCAGTTAAAAATATTATGYTAGGACACGCTAGTGTGCAAGCAGAAATGGAACAAGTTGTGGARGTDTATGAATATGCCCAAAAATTGGGTGGAGAAGCAGGATTCTACCATATATTGAAYAACCCAAAAGCATCATTATTATCTTTGACTCAATTYCCYCACTTCTCYAGTGTAGTATTRGGCAATGCTGCTGGCCTAGGCATAATGGGAGAATACAGAGGTACACCAAGGAATCAAGATCTATATGATGCTGCDAARGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGTGTATTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAACATCAGCTTAATCCAAAAGATAATGATGTAGAGCTTTGA RSV B1 N ORF consensus (SEQ ID NO: 33)ATGGCTCTTAGCAAAGTCAAGTTRAATGATACATTAAATAAGGATCAGCTGCTGTCATCCAGCAAATACACTATTCAACGTAGTACAGGAGATAATATTGACACTCCCAATTATGATGTRCAAAAACACYTAAACAAACTATGTGGTATGCTATTAATCACTGAAGATGCAAATCATAAATTCACAGGATTAATAGGTATGYTATATGCTATGTCCAGGTTAGGAAGGGAAGACACTATAAAGATACTTAAAGATGCTGGATATCATGTTAAAGCTAATGGAGTAGATATAACAACATATCGTCAAGATATAAATGGAAAGGAAATGAAATTCGAAGTATTAACATTATCAAGCTTGACATCAGAAATACAAGTCAATATTGAGATAGAATCTAGAAAGTCCTACAAAAAAATGCTAAAAGARATGGGAGAAGTGGCTCCAGAATATAGGCATGAYTCTCCAGACTGTGGGATGATAATACTRTGTATAGCWGCHCTTGTVATAACCAAATTAGCAGCAGGAGAYAGATCAGGTCTYACAGCAGTAATTAGGAGRGCRAACAATGTCTTAAAAAACGAAATAAAACGHTACAAGGGCCTVATACCAAAGGAYATAGCYAACAGTTTTTATGAAGTGTTTGAAAAACACCCTCATCTTATAGATGTTTTYGTGCACTTTGGCATTGCACAATCATCCACAAGAGGGGGTAGTAGAGTTGAAGGAATCTTTGCAGGATTRTTTATGAATGCCTATGGTTCAGGRCAAGTAATGCTAAGATGGGGAGTTTTAGCCAAATCTGTAAAAAATATCATGCTAGGWCATGCTAGTGTCCARGCAGAAATGGAGCAAGTTGTDGAAGTCTATGAGTATGCACAGAAGTTGGGRGGAGAAGCTGGWTTCTACCATATATTGAACAATCCAAAAGCATCATTGCTGTCATTAACTCAATTTCCYAACTTCTCAAGTGTGGTCCTAGGCAATGCAGCAGGYCTAGGCATAATGGGAGAGTATAGAGGTACACCAAGAAACCARGATCTYTATGATGCWGCCAAAGCATATGCAGAGCAACTCAAAGAAAATGGAGTAATAAACTACAGTGTATTAGACTTAACARCAGAAGAATTGGAAGCCATAAAGMATCAACTCAACCCCAAAGAAGATGAYGTAGAGCTYTAA

Exmaple 3 Determination of Genotype Using Quantitative Real-Time PCR(qRTPCR) Assay

Primers that specifically amplify the N region of both the RSV A2 andthe RSV B1 subtypes were designed using the consensus sequencesdescribed. The sequences for these primers (RSV499 and RSV636) are setforth below in Table 1. Alternate primers for specifically amplifyingthe N region of both RSV A and RSV B subtypes are RSV322 and RSV458.

A probe that specifically binds to an RSV A nucleic acid sequence(RSVA576) and a probe that specifically binds to an RSV B (RSVB576)nucleic acid sequence also were designed. Alternate probes forspecifically binding RSV A nucleic acid sequences or RSV B nucleic acidsequences are RSVA349 and RSVB349, respectively. The sequences for theseprobes are shown in Table 1.

For use as controls, a primer pair that amplifies an M2 sequence (MS2Fand MS2R) and a probe that specifically binds to an M2 nucleic acidsequence were designed. The sequences for these primers and this probeare shown in Table 1.

TABLE 1 Sequences for RSV and MS2 (recovery control) primers and probesRSV499 GTV ATA ACC AAA TTA GCA GC (SEQ ID NO: 1) RSV636CAC TTC ATA RAA RCT GTT DGC (SEQ ID NO: 2) RSV322RGAAATGAAATTYGAAGTRTTAAC (SEQ ID NO: 34) RSV458GARTCATGCCTRTATTCTGGAGC (SEQ ID NO: 35) RSVA576VIC/TGA AAT GAA ACG TTA TAA AGG CTT AC/BHQ (SEQ ID NO: 3) RSVB576FAM/CGA AAT AAA ACG HTA CAA GGG CCT VA/ZEN (SEQ ID NO: 4) RSVA349VIC/GCAAGCTTAACAACTGAAATTCAAATCAAC/BHQ (SEQ ID NO: 36) RSVB349FAM/TCAAGCTTGACATCAGAAATACAAGTCAAT/ZEN (SEQ ID NO: 37) MS2FTGT GGA GAG ACA GGG CAC TG (SEQ ID NO: 30) MS2RCAG TTG TTG GCC ATA CGG ATT (SEQ ID NO: 31) MS2probeNED/TAA GGC CCA AAT CTC AGC CAT GCA TC/BHQ (SEQ ID NO: 5)

The materials that were used for the viral load quantification includedRSV RNA samples, primer and probes (10 μM each), and ThermoFisherAg-Path ID One-Step RT-PCR (Catalogue No. AM1005M).

The RSV load quantification was performed as follows. The RNA templates,primers, probes, and 2× reaction mix were thawed at room temperature andimmediately placed on ice once thawed. Enzyme was kept at −20° C. untilready to use. The RT-PCR mastermix was prepared on ice using the samplevolume below:

Volume Final concentration 2 × reaction mix 12.5 μl Forward primer-RSV0.5 μl 200 nM Reverse primer-RSV 0.5 μl 200 nM Forward primer-MS2 0.25μl 100 nM Reverse primer-MS2 0.25 μl 100 nM Probe-RSVA/VIC 0.25 μl 100nM Probe-RSVB/FAM 0.5 μl 200 nM Probe-MS2/NED 0.25 μl 100 nM Water 7.5μl 25 × enzyme mix 1.5 μl Total 25 μl (when the RNA template 1 μl isadded)

The required mastermix was aliquoted into each PCR tube. One μl of RNAtemplate was added into its designated tube, and water was used as anegative control. The lids were closed and the tubes quickly spun tocollect all liquids. The tubes were placed in the thermocycler, and thefollowing reaction conditions were used:

RT reaction (cDNA synthesis) 50° C. 10 min RT inactivation 95° C. 10 minPCR 40 cycles Denature 95° C. 15 sec Anneal 45° C. 45 sec

The RT-PCR amplicon resulting from amplification with primers RSV499 andRSV636 was about 137 bp. The qRT-PCR assay provided the desiredsensitivity in the 7 log₁₀ dynamic range; Lower limit of detection(LLOD) ˜100 copies. The assay also provided desired subtype specificity.As shown in FIG. 1, RSV A and RSV B are detectable and quantifiable in amultiplexed reaction.

To determine whether the qRT-PCR assay was able to provide genotypingresults comparable to commercially available qualitative assays, testingof RSV samples was performed with either the present qRT-PCR assay orwith the GenMark Diagnostics RVP qualitative assay. The results of thecomparative study with GenMark Diagnostics RVP qualitative assay areshown in FIG. 2. The data shown in the tables is the result of reactionsthat were performed in triplicate. The left table shows the quantitativeresults using the qRT-PCR assay with two known RSV A samples (PC1_A andPC2_A), two known RSV B samples (PC1_B and PC2_B), and two controls thatdid not include a template. The numbers shown are the Ct values, whichindicate the copy number present in the sample. UND indicates that theparticular RSV type was not detected in the reaction. The data in theright two tables shows qualitative results of the qRT-PCR assay and theGenMark RVP assay. Notably, one RSV sample was able to be genotyped bythe present qRT-PCR assay, but was not detected or typed by the GenMarkRVP assay.

These results show that a multiplex RSV A and RSV B specific viral loadand typing assay that includes a recovery control was successfullydeveloped. The accuracy of RSV typing was confirmed based on concordancewith an FDA-cleared test as well as F gene sequencing.

A proficiency panel was conducted to assess the sensitivity of themultiplex qRT-PCR assay, and the results are shown in FIG. 3. The figurepresents tables showing data that are the result of triplicate samplesof known viral load, reported in copies/mL. Known VL refers to the knownviral load as detected using a singleplex assay, and MGB 3plex refers tothe assay in which the subtype A probes, subtype B probes, and controlprobes are included in the same reaction. The data demonstrates thatthere is no loss in sensitivity in the multiplexed reaction.

Exmaple 4 Quantitation of Viral Load

Commercially available RSV kits thus far have only been qualitative. Asset forth in the following example, an RSV triplex qRT-PCR assay wasdeveloped that quantifies RSV viral load (copies/ml), subtypes for RSVAB strains and is formatted to detect an internal control. The accuracyof the quantitation of the qRT-PCR assay was analyzed using an in vitrotranscript (IVT) control. The RT-PCR amplicon resulting from primersRSV499 and RSV636 described above was cloned into a plasmid. Theamplicon was gel purified and cloned according to the manufacturer'sprotocol (ThermoFisher, Catalogue No. K4575-02) into pCR®4-TOPO (FIG. 4)to create the pCR4-TOPO-RSVA2.N plasmid and the pCR4-TOPO-RSVB1.Nplasmid. These vectors were used in an in vitro transcription assay,along with the ThermoFisher MEGAscript T7 Transcription kit (CatalogueNo. AM1334), the ThermoFisher MEGAclear kit (Catalogue No. AM1908), andNew England BioLabs (Ipswich, Mass.) SpeI restriction enzyme.

The DNA concentration of each plasmid was measured, and 10 μg of eachplasmid was linearized using the following digestion conditions:

10 μg DNA x μl SpeI 5 μl Cutsmart buffer 10 μl Water y μl Total 100 μl

Linearization was verified on a 1% agarose gel. The gel purificationprotocol using QlAquick spin columns (Qiagen) was followed. At theelution step, the elution was performed with 23 μl RNAse free ddH₂O fromthe MEGAscript T7 transcription kit (optional: prewarmed to 55° C.). TheMEGAscript T7 kit reagents (10× reaction buffer, ribonucleotides) werethawed, and T7 enzyme was kept at −20° C. The nucleotide and reactionbuffer mastermix was prepared per sample volume below:

ATP solution 2 μl CTP solution 2 μl GTP solution 2 μl UTP solution 2 μl10 × reaction buffer 2 μl

Then 10 μl of the mixture was distributed into tubes, and 8 μl oflinearized DNA template and 2 μl of enzyme were added. The mix waspipetted and subjected to centrifugation if necessary. The reaction wasincubated at 37° C. for 2 hours. One μl of TURBO DNAse mix was added,and the reaction was incubated for 15 minutes at 37° C. Meanwhile, aportion of the elution solution was heated to 95° C. Then 80 μl ofelution solution (room temp) was added to each tube and mixedthoroughly.

Then 100 μl of sample was transferred to 350 μl of Binding Solutionconcentrate and mixed thoroughly. Ethanol (250 μl of a 100% solution)from a new bottle was added to each sample and mixed gently. Then 700 μlof the sample was added to a column (provided in kit) and spun at 13,000RPM. The flow-through was discarded. The column was washed twice with500 μl Wash Solution and spun for 4 minutes after the last wash toremove all traces of Wash Solution. Then 50 μl of Elution Solution (roomtemp) and 50 μl Elution Solution (95° C. heated) were added andincubated in the column for 1 minute. Elution was performed by spinningat 13,000 RPM into a new collection tube. Eluted RNA was stored at −80°C. Quality of the RNA was assessed (Agilent RNA 6000 Nano kit, CatalogueNo. 5067-1511), and the yield was determined (ThermoFisher NanoDrop2000c)(Palo Alto, Calif.). The RNA was approximately 220 nucleotides(nt) in length.

Copy Number Calculation

The number of RNA copies was determined using the following formula onthe online calculator set forth below:copies=(ng*6.022×10²³/mole)/(length*1×10⁹ ng/g*325 g/mole) The onlinecalculator can be found at: http://www.endmemo.com/bio/dnacopynum.php.

The amount of purified RNA was measured on a ThermoFisher NanoDrop 2000cand determined to be 499 ng/μl for the RSV A2 in vitro transcript (IVT)and 584 ng/μl for the RSV B1 IVT.

The following information was entered into the online calculator usingthe input screen shown in FIG. 5:

Choose option ‘ssRNA’

Sequence length=200 nt

Weight=499 ng

Click ‘calculate’

A dilution series was prepared as shown below:

(4.2×10¹² copies/μl)(x)=(5×10¹⁰ copies/μl)(100 μl)

x=(5×10¹⁰ copies/μl)/(4.2×10¹² copies/μl)

x=1.2 μl RSV A2 IVT+98.8 μl RNAse-free ddH₂O

Then, serial 10-fold dilutions were made (for 1×10², make 1:5 of 5×10²).The calculations and serial dilutions were repeated for RSV B1 IVT.

Exmaple 5 Sequencing of RSV F ORF

The RSV A and B sequences were amplified using reverse transcription PCR(RT-PCR). The RSV A2 reference sequence can be found under GenBankAccession No. JX198138. The RSV B1 reference sequence can be found underGenBank Accession No. AF013254. Primers for reverse transcription andamplification of RSV A or RSV B were designed and are shown in Table 2.

TABLE 2 RSV F ORF reverse transcription primers: 5_RSV_A2GGG CTC GAG ACC GGT TCT GGG GCA AAT AAC AAT GG (SEQ ID NO: 6) PCR primer3_RSV_A2GGG TCT AGA ACG CGT TAG GTG CTA TTT TTA TTT AGT TAC (SEQ ID NO: 7)RT and/or PCR primer 5_RSV_B1GGG CTC GAG ACC GGT CCT GGG GCA AAT AAC CAT GG (SEQ ID NO: 8) PCR primer3_RSV_B1GGG TCT AGA ACG CGT TCA GGT GGT TTT TTG TCT ATT TGC (SEQ ID NO: 9)RT and/or PCR primer

The RNA samples were used in an RT-PCR assay using a 5′ and 3′ primerset (either A2 or B1) (10 μM each) and the ThermoFisher SuperScript IIIKit with Platinum Taq (Catalogue No. 12574-026) (Waltham, Mass.).

The protocol for the RT-PCR of the RSV isolates included the followingsteps. RNA templates, primers, and 2x reaction mix were thawed at roomtemperature and immediately placed on ice once thawed. Enzyme was keptat −20° C. until ready to use. The RT-PCR mastermix was prepared on iceusing per sample volume below:

2 × reaction mix 25 μl Forward primer 1 μl Reverse primer 1 μl Water 17μl SSIII/Taq 1 μl RNA template 5 μl Total 50 μl

The required mastermix was aliquoted into each PCR tube. The amount ofRNA template was added into its designated tube, and water was used as anegative control. The lids were closed and the tubes were spun quicklyto collect all liquids. The tubes were then placed in the thermocycler,and the reactions were run as follows:

RT reaction 55 C 30 min RT inactivation 94° C. 2 min PCR 40 cyclesDenature 94° C. 30 sec Anneal 55° C. 30 sec Extend 68° C. 3 min Finalextension 68° C. 5 min Storage  4° C. indefinite

The resulting RT-PCR amplicons were approximately 2800 bp. They were runon a 1% agarose gel, excised, and gel purified using the QlAquick gelextraction kit (Qiagen, Valencia, Calif., Catalogue No. 28704).

The purification included the following steps. Three volumes of BufferQG were added to 1 volume of gel (100 mg=100 μl). The gel was incubatedin the buffer at 50° C. for 10 minutes. After the gel had dissolved, thecolor of the mixture was checked to confirm it was yellow (similar toBuffer QG without dissolved agarose). One gel volume of isopropanol wasadded to each sample and mixed. DNA was bound into a QIAquick spincolumn and centrifuged at 13000 rpm for 1 minute. Flow-through wasdiscarded. Columns were washed by adding 0.75 mL of Buffer PE andcentrifuged for 1 minute. Flow-through was discarded, and the emptycolumn was centrifuged for an additional minute. The QlAquick spincolumn was placed into a clean Eppendorf tube, the DNA was eluted in 40μl Buffer EB, and the concentration of DNA was measured.

Sequencing primers were designed against A2 and B1 reference strains.Ten primers each were tested for the sequencing of the F open readingframe of RSV subtypes A and B. Forty-five RSV samples and 2 referenceRSV strains were analyzed using the designed primers. The generalposition of the tested primers is shown in FIG. 5A and a neighborjoining tree of RSV A and RSV B samples tested in RSV sequencing assayis shown in FIG. 5B.

Tables 3 and 4 show the primers that were used for Sanger sequencing.

TABLE 3 RSV A2 F ORF sequencing primers: 440F.RSV.ENVS.A2CTC TGG GGC AAA TAA CAA TGG AGT TG (SEQ ID NO: 10) 74F.RSV.ENVS.A2GTC AAA ACA TCA CTG AAG AAT TTT ATC (SEQ ID NO: 11) 407R.RSV.ENVS.A2CTT CTT TTC CTT TTC TTG CTT AAT G (SEQ ID NO: 12) 449R.RSV.ENVS.A2CTG GCG ATT GCA GAT CCA ACA CCT A (SEQ ID NO: 13) 508F.RSV.ENVS.A2GCT CTA CTA TCC ACA AAC AAG GCT GTA GTC (SEQ ID NO: 14) 792F.RSV.ENVS.A2GCC TAT AAC AAA TGA TCA GAA AAA G (SEQ ID NO: 15) 867R.RSV.ENVS.A2CAT GAT AGA GTA ACT TTG CTG TCT AAC T (SEQ ID NO: 16) 1043R.RSV.ENVS.A2GAT CCT GCA TTG TCA CAG TAC CAT CC (SEQ ID NO: 17) 1480R.RSV.ENVS.A2GAG ATA TTG ATG CAT CAA ATT CAT C (SEQ ID NO: 18) 1397F.RSV.ENVS.A2GTC TCT ATG TAA AAG GTG AAC CAA TA (SEQ ID NO: 19)

TABLE 4 RSV B1 F ORF sequencing primers: 33R.RSV.ENVS.B1GAT TGC ACT TGA TCT ATG GAT CAG C (SEQ ID NO: 20) 74F.RSV.ENVS.B1GTC AGA ACA TAA CTG AGG AGT TTT ACC (SEQ ID NO: 21) 484R.RSV.ENVS.B1CTT CAA GGT GTA GAA CTT TGG ATA CAG (SEQ ID NO: 22) 664F.RSV.ENVS.B1GAA TTC CAG CAG AAG AAC AGC AGA TTG (SEQ ID NO: 23) 835F.RSV.ENVS.B1CAG ATA GTA AGG CAA CAA AG (SEQ ID NO: 24) 901F.RSV.ENVS.B1GTA CAG CTA CCT ATC TAT GG (SEQ ID NO: 25) 1104R.RSV.ENVS.B1GTC ACA AAA TAC TCG ATT GGA C (SEQ ID NO: 26) 1174R.RSV.ENVS.B1CAT ACT TGG AAT TGA ATA TGT CAG (SEQ ID NO: 27) 1121F.RSV.ENVS.B1CAT TAC CAA GTG AAG TCA GCC TTT G (SEQ ID NO: 28) 1324F.RSV.ENVS.B1GTG TCA AAC AAA GGA GTA GAT ACT G(SEQ ID NO: 29)

The following materials were used in the sequencing reaction: Gelpurified DNA samples, Big Dye, Dilution Buffer, Sequencing primers (seetables above) (10 μM each), SAM solution, and BDX beads.

Sequencing mastermix was prepared on ice using per sample volume below:

Dilution buffer 1.5 μl Big Dye v3 1 μl Water 5.5 μl Primer 1 μl Total 10μl (after adding 1 μl of DNA template (~40 ng)

Nine μl of mastermix were transferred to individual PCR tubes (orskirted 96 well plate), and 1 μl of DNA template was added toappropriate wells. The tubes were placed in the thermocycler, and thereactions were run as follows:

Initial denaturation 96° C.  2 min PCR 35 cycles Denature 96° C. 10 secAnneal 50° C.  5 sec Extend 60° C.  4 min Storage  4° C. indefinite

Following the PCR reaction, 1.1 mL BDX beads and 5.1 mL SAM solutionwere combined and periodically vortexed to keep the beads in suspension.Then 55 μl of mixture was dispensed into to each PCR tube. The plate wassealed and shaken for 20 minutes at 4000 RPM. The plate was spun for 2minutes at 1000 RPM. The sequencing samples were run in an ABI machineusing GenSeq software.

Alternatively, the F glycoprotein sequences of cultured hRSV isolateswere amplified by RT-PCR, and the samples were subjected to AxyPrep®magnetic PCR purification, Nextera DNA library preparation, and MiSeq®paired end sequencing. Analysis of the reads was performed using a deepsequencing pipeline. FIG. 6A shows a quality control plot for IlluminaMiSeq sequencing of an hRSV isolate, showing that the Q-score is greaterthan 30 across reads. FIG. 6B is a quality control plot showing thatgreater than 99% of reads mapped to the reference genome with evenlydistributed read and insert lengths. Minor variants were detected using3-10% thresholds, with accompanying data on each variant codon, aminoacid, and frequency.

FIG. 7A is a schematic diagram showing the relative locations of thesequencing primers used to sequence the RSV F ORF. FIG. 7B is a neighborjoining tree of the RSV A and RSV B samples tested in the RSV sequencingassay, generated using the sequence data obtained from the sequencingmethods above.

What is claimed is:
 1. A method for simultaneously determining the viralload and genotype of a respiratory syncytial virus (RSV) in a biologicalsample, comprising: a) amplifying a nucleic acid sequence encoding anRSV open reading frame (ORF) in a biological sample using a set ofprimers that specifically amplifies an RSV A ORF and/or an RSV B ORF andat least two nucleic acid probes in a quantitative polymerase chainreaction assay, wherein at least one probe specifically binds to an RSVsubtype A nucleic acid sequence and at least one probe specificallybinds to an RSV subtype B nucleic acid sequence, and wherein the atleast two nucleic acid probes are differentially labeled; b) determiningthe viral load and the RSV subtype(s) present in the biological sample.2. The method of claim 1, wherein amplifying the nucleic acid sequencecomprises reverse transcription and real-time PCR amplification.
 3. Themethod of claim 2, wherein reverse transcription and real-timeamplification occur in a single step.
 4. The method of any of claims 1,wherein the set of primers amplifies a region of the RSV nucleocapsid(N) protein in RSV A and/or RSV B.
 5. The method of any of claims 1,wherein the set of primers comprises a nucleic acid sequence comprisingSEQ ID NO:1 and a nucleic acid sequence comprising SEQ ID NO:2.
 6. Themethod of any of claims 1, wherein the probe that specifically binds toan RSV subtype A nucleic acid sequence comprises SEQ ID NO:3 and theprobe that specifically binds to an RSV subtype B nucleic acid sequencecomprises SEQ ID NO:4.
 7. The method of any of claims 1, furthercomprising a set of primers that amplifies an MS2 region and a nucleicacid probe that specifically binds to a M2 nucleic acid.
 8. The methodof claim 7, wherein the set of primers comprises a nucleic acid sequencecomprising SEQ ID NO:30 and a nucleic acid sequence comprising SEQ IDNO:31.
 9. The method of claim 7, wherein the nucleic acid probe thatspecifically binds to a M2 nucleic acid sequence comprises SEQ ID NO:5.10. The method of any of claims 1, wherein the primers and/or probes arelabeled with a fluorescent moiety.
 11. A method for determining thegenotype or viral load of a respiratory syncytial virus (RSV) in abiological sample, comprising: a) amplifying a nucleic acid sequenceencoding an RSV open reading frame (ORF) in a biological sample using aprimer that comprises SEQ ID NO:1; a primer that comprises SEQ ID NO:2;and a probe that comprises SEQ ID NO:3, a probe that comprises SEQ IDNO:4, or both a probe that comprises SEQ ID NO:3 and a probe thatcomprises SEQ ID NO:4 in a quantitative polymerase chain reaction assay,wherein the probes are differentially labeled; and b) determining thepresence or absence of an amplification product that binds one of theprobes, or determining the viral load, thereby determining the RSVgenotype(s) present or viral load of the RSV in the biological sample.12. The method of claim 11, wherein amplifying the nucleic acid sequencecomprises reverse transcription and real-time PCR amplification, andwherein reverse transcription and real-time amplification occur in asingle-step.
 13. The method of claim 11, wherein a probe that comprisesSEQ ID NO:3 or a probe that comprises SEQ ID NO:4 is used.
 14. Themethod of claim 11, wherein both a probe that comprises SEQ ID NO:3 anda probe that comprises SEQ ID NO:4 are used.
 15. The method of any ofclaims 11, further comprising a set of primers that amplifies an MS2region and a nucleic acid probe that specifically binds to a M2 nucleicacid.
 16. The method of claim 15, wherein the set of primers comprises anucleic acid sequence comprising SEQ ID NO:30 and a nucleic acidsequence comprising SEQ ID NO:31.
 17. The method of claim 15, whereinthe nucleic acid probe that specifically binds to a M2 nucleic acidsequence comprises SEQ ID NO:5.
 18. The method of any of claims 11,wherein the primers and/or probes are labeled with a fluorescent moiety.19. A nucleic acid probe comprising SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:36, SEQ ID NO:37, or SEQ ID NO:5; and a detectable moiety.
 20. Thenucleic acid probe of claim 19, wherein the detectable moiety is afluorescent moiety.